Flow cytometry is a well accepted tool in research. It allows the user to analyze or sort tens of thousands of cells at rates of up to 10,000 cells per second. The power of this tool continues to expand and more recently applications have been designed and approved for using flow cytometers as clinical instruments in the evaluation and characterization of diseases.
An in-depth description of how a flow cytometer operates can be found in a number of references. Herzenberg et al., Sci. Amer., 231:108 (1976), describe the operation and arrangement of the prototypic instrument. Essentially, the instrument is designed to take a sample of cells and pass the cells, substantially one at a time, through a zone of illumination, wherein each cell is illuminated by a light source, typically a light source of a single wavelength such as a laser, and light scattered by each cell is collected by a series of light detectors. The results are stored in a data storage means, such as a computer, for analysis. In another form, the cells can be separated based upon their optical and other characteristics as they exit the zone of illumination in order that they may be used in further experimentation. Two examples of flow cytometers are set forth in U.S. Pat. Nos. 4,284,412 and 3,826,364.
In order to get the cells to flow through the zone of illumination substantially one at a time, the cells in the sample pass through a nozzle assembly (see e.g., 12 in FIG. 1 of U.S. Pat. No. 3,826,364) in a liquid buffer and then are coaxially fed through the zone in a particle-free sheath fluid. If the cells are to be separated, the nozzle can be vibrated at specific frequencies to cause drops to form at the tip of the nozzle wherein each drop contains a single cell.
The data collected from each cell falls into two types: scatter properties and immunofluorescent properties. In the former, each cell will scatter light from the light source based upon the size of the cell and its granularity. Scattered light can be collected by means of photodetectors which are placed at certain angles to the light source. Immunofluorescence, on the other hand, is a function of having labelled the cells with one or more immunofluorescent markers, such as a monoclonal antibody conjugated to fluorochrome. In this case, the fluorochrome(s) must be excitable by the light source and must emit at wavelengths that have peaks which do not overlap. Thus, by labelling the cells in the sample with one or more immunofluorescent markers, both scatter and immunofluorescence can be used to identify a particular cell. U.S. Pat. Nos. 4,559,307 and 4,607,007 provide two examples of how scatter and immunofluorescence can be used together to discriminate between cells in a sample.
In a typical experiment, 10,000 to 200,000 cells are analyzed. Between experiments, most manufacturers recommend that the sample buffer be introduced into the sample port and run through the instrument to clean out the entire system, including the nozzle (or flow cell). Using this method, however, it has been found that up to 0.1% of the cells in the sample remain in the system and are not washed out by routine cleaning. Generally, this does not present a problem; however, if one is looking for rare events in a sample (i.e., events that occur on the order of 1:1,000,00), then a 0.1% contamination from prior samples may lead to spurious results.
Accordingly, what is needed is a method to clean the fluidics systems of a flow cytometer which reduces the contamination in the system to essentially zero.